PROJECT SUMMARY/ABSTRACT Insomnia is highly prevalent in adolescence, particularly in post-pubertal girls and tends to persist over time. Insomnia is considered a hyperarousal disorder, in which abnormally elevated levels of cognitive and physiological activation, particularly evident at bed-time, prevent individuals from falling asleep and having a restorative night?s sleep. Hyperarousal is a major pathophysiological mechanism linking insomnia with poor mental and physical health, including cardiovascular (CV) disease. We and others have shown evidence of autonomic nervous system (ANS) dysfunction such as hyperactivation of the sympathetic branch of the ANS ? a major etiological factor in CV disease ? in young and midlife adults, both before and during sleep. It is unknown if ANS hyperarousal is evident in adolescents with insomnia. The biological basis for an emerging sex difference in insomnia prevalence in adolescence is unknown. Our pilot data reveal intriguing evidence of sex-differences in basic and stress-dependent ANS modulation during sleep in adolescents with girls showing a greater ANS response to stress. These data suggest the existence of a predisposing and stress-dependent ANS vulnerability in female adolescents, a potential pathway to develop insomnia. This proposal takes a novel approach to investigating the manifestation of physiological ANS and CV hyperarousal in adolescents with insomnia by experimentally manipulating the pre-sleep arousal state via stress-induced ANS up-regulation and relaxation-driven ANS down-regulation. In addition, the proposal focuses on sex differences in ANS and CV responses to pre-sleep ANS manipulation, potentially addressing the question of why female sex is a major risk factor for insomnia. We aim to test 110 male and female high-school students (16-18y) with and without DSM-5 Insomnia Disorder, during a regular in-lab polysomnographic night (baseline) and under experimental pre-sleep stress (psychosocial stressor) and pre-sleep relaxation (Virtual reality ANS bio-feedback) intervention nights, using state-of-the-art, noninvasive, beat-to-beat ANS and CV measures, including blood pressure, to assess nocturnal ANS and CV function in adolescents with insomnia (Aim 1); the impact of pre-sleep ANS arousal levels on nocturnal ANS and CV function, and sleep in adolescents with and without insomnia, considering possible sex differences (Aim 2), and the extent to which nocturnal ANS and CV function mediate the effect of pre-sleep arousal levels on objective and perceived sleep quality (Aim 3). This proposal has the potential to elucidate pathophysiological ANS hyperarousal underlying Insomnia Disorder in adolescence, including potential reasons for the vulnerability to insomnia in girls, leading to better recognition and potentially new treatment strategies of this disorder targeted at the state of ANS hyperarousal in the pre-sleep period.